The EBV membrane antigen (EBV-MA) gp350 contains virus neutralizing epitopes and is currently being tested as a possible EBV vaccine candidate in England. A series of monoclonal antibodies (mAbs) to identifiably different epitopes on EBV-MA was obtained from Louis Qualtiere. Several additional mAbs were obtained from Gary Pearson. These have been used to screen antigen expressing clones for reactivity to different epitopes in order to identify the nucleic acid sequences important for virus neutralization. A clone of the EBV Bam L fragment which contains the coding sequence for the gp350 was used with a bacterial expression vector, pWS50, to generate ten overlapping clones which express different portions of unglycosylated gp350. The antigens produced by eight of these clones were purified and used in a dot blot immunoassay with the mAbs to map the epitopes on EBV-MA. Four of fifteen mAbs tested reacted with the recombinant E.coli antigens, and three antigenic epitopes (nucleotides 1980-2307, 3186-3528 and 3528-3576 in Bam HI L) identified. The remaining mAbs may only recognize the glycosylated form of gp350. Proteins expressed by four of the clones were used to produce antisera in rabbits. When tested for neutralization of EBV in tissue culture, the rabbit antisera as well as the mAbs which recognized the three epitopes failed to neutralize, suggesting that these epitopes are not involved in neutralization. A manuscripts describing these data has been accepted for publication in J Gen Virol. The entire gp350 coding sequence has also been expressed in a Baculovirus expression system which glycosylates the protein. The properties of the Baculovirus-expressed protein, including its glycosylation and reactivity with mAbs, are being evaluated. Subclones which express different portions of gp350 in a glycosylated form in the Baculovirus expression system are currently being prepared for additional epitope mapping studies.